1. Field of the Invention
This invention relates to a gene, the Or gene, cloned from an orange cauliflower mutant, a construct containing the gene and its promoter, a vector and method of transforming plants utilizing the construct and vector, and plants, including staple crops, transformed with the gene construct.
2. Description of the Relevant Art
Carotenoids are a diverse group of pigments widely distributed in nature. They are synthesized de novo in all photosynthetic organisms, as well as in some non-photosynthetic bacteria and fungi (Goodwin and Britton. 1988. In: Plant Pigments, Goodwin, T. W., ed., Academic Press, London, pages 61-132). Carotenoids fulfill many essential functions in plants (Demmig-Adams and Adams. 1996. Trends Plant Sci. 1: 21-26; Frank and Cogdell. 1996. Photochem Photobiol. 63: 257-264; Yamamoto et al. 1997. Photochem Photobiol. 65: 62S; Niyogi, K. K. 1999. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 333-359; Ma et al. 2003. Proc. Natl. Acad. Sci. USA 100: 4377-4382). They play important roles in human nutrition and health as the primary dietary precursor of vitamin A that fulfills many physiological functions in humans such as vision, reproduction, and cell proliferation (Combs, G. F. Jr. 1998. The Vitamins: Fundamental Aspects in Nutrition and Health, 2nd Edition, Academic Press, San Diego) and in reducing risks of cancer and other chronic diseases (Mayne, S. T. 1996. FASEB J. 10: 690-701; Smith-Timothy, A. D. 1998. British J. Biomed. Sci. 55: 268-275; Giovannucci, E. 1999. J. Natl. Cancer Inst. 91: 317-331; Bertram, J. S. 1999. Nutr. Rev. 57: 182-191; Palace et al. 1999. Free Radical Biol. Med. 26: 746-761).
Carotenoid biosynthesis is a multifaceted and highly regulated process in plants (Hirschberg, J. 2001. Curr. Opin. Plant Biol. 4: 210-218; Bramley, P. M. 2002. J. Exp. Bot. 53: 2107-2113). Exciting progress has been made in identifying and characterizing genes encoding enzymes for the biosynthesis of carotenoids in plants (Cunningham and Gantt. 1998. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 557-583; Hirschberg, supra; Fraser and Bramley. 2004. Prog. Lipid Res. 43: 228-265). Comparatively, little is known about the regulatory mechanisms underlying carotenoid accumulation and/or the signal pathways that trigger the differentiation of plastids into chromoplasts.
To gain new insights into the molecular control of carotenogenesis in plants, we are studying the Or gene in cauliflower (Brassica oleracea var. botrytis). The cauliflower Or gene arose as a result of spontaneous mutation. It causes many low-pigmented tissues of the plant, most noticeably the edible curd and shoot, to accumulate carotenoids (Crisp et al. 1975. Euphytica 24: 173-176; Dickson et al. 1988. Hortscience 23: 778-779; Li et al. 2001. Plant J. 26: 59-67) (FIG. 1). Our previous studies showed that the predominant carotenoid that accumulated in the affected tissues is beta-carotene, which can reach levels of several hundred fold higher than those found in the comparable tissues of wild type cauliflower (Li et al., supra). The Or gene appears not to exert its effect via the direct regulation of carotenogenic gene expression. Therefore it represents a novel carotenoid gene mutation. Such carotenoid mutants can provide useful tools for exploring the regulation of carotenoid accumulation.
The indispensable role of carotenoids in plants and the increasing interest in their health benefits to humans have prompted a significant effort to understand carotenoid biosynthesis in plants. Although major genes and their biochemical roles in carotenogenesis have been well documented, the control mechanisms regulating the overall carotenoid biosynthesis remain an enigma (Cunningham and Gantt, Hirschberg Fraser and Bramley, supra). Only a few gene mutations are known to induce carotenoid accumulation in unpigmented tissues (Crisp et al., supra; Buishand and Gabelman. 1979. Euphytica 28: 611-632). The high-beta-carotene Or (Orange) gene mutation in cauliflower provides us an ideal model to divulge the regulatory control as it switches on carotenogenesis in tissues where the activity of this pathway is normally repressed (Li et al., 2001, supra).
Vitamin A deficiency is one of the most widespread nutrient deficiencies, that affects approximately 400 million people in developing countries due to low levels of carotenoids in their diets. Vitamin A deficiency results in blindness, poor immune function, and early death. Because of the important role of carotenoids in plants and their beneficial effects for humans and animals, knowing the determinants of carotenoid accumulation could provide a novel and powerful tool, along with the catalytic genes, to enrich carotenoids in important food crops. Biofortification of staple crops with carotenoids is considered to be a very effective and sustainable approach to help afflicted populations fight against vitamin A deficiency. In addition, there has been increasing public interest in using carotenoids as antioxidants or nutraceutical supplements to reduce risks of cancer and cardiovascular disease, and to protect against age-related eye diseases such as macular degeneration, the leading cause of age-related blindness in the world. The Or gene could be used to alter food plants to selectively modify carotenoid content and/or composition to provide protection against these diseases, thus improving various aspects of human well-being and reducing the economic costs associated with these diseases. Additional uses for the Or gene include its use to impart novel color variation in plants, and to provide feed additives in animal feeds. Therefore, it is a primary object of this invention to provide a molecular tool and method for increasing accumulation of carotenoids in cells as development of carotenoid-enriched plant foods will be the most effective approach to maximize the nutritional and health benefits of carotenoids.